The innate immune response plays a central role in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury. However, the mechanisms are unclear. Toll-like receptor (TLR) mediated signaling plays a critical role in the induction of the innate immune response. During the last grant period, we demonstrated that TLR4 mediates myocardial injury in response to I/R. We discovered that PI3K/Akt signaling may be a negative feedback regulator of the TLR4 response. Our data suggest that differential regulation of TLR and PI3K/Akt pathways determines the fate of the myocardium in response to ischemic insult. In the current proposal, we will investigate the differential regulation and interplay between TLRs and PI3K/Akt signaling during myocardial I/R. Our long term goals are to elucidate the mechanisms of cardiac ischemic injury and to develop effective approaches to prevent myocardial I/R injury. Our hypothesis is: Differential regulation of TLR and PI3K/Akt signaling pathways determines the fate of the myocardium in response to ischemia/reperfusion insult. Specific Aim 1 will define the mechanisms by which TLR4 mediates cardiomyocyte apoptosis following I/R. We found that TLR4 activation in I/R directly contributes to myocardial apoptosis. We will determine whether TLR4 will stimulate apoptotic signaling pathways following myocardial I/R. Specific aim 2 will elucidate the mechanisms of TLR4 negative regulation of PI3K/Akt activity during myocardial I/R. TLR4 plays a major role in the pathology of cardiac I/R. PI3K/Akt dependent mechanisms attempt to inhibit the deleterious effects of TLR4 signaling in I/R. We will define how TLR4 negatively regulates PI3K/Akt signaling in cardiac I/R injury, thereby limiting its beneficial effect. Specific aim 3 will examine the mechanism by which TLR2 activates PI3K/Akt, resulting in protection of the myocardium from I/R. We found that stimulation of TLR2 induces cardioprotection through PI3K/Akt dependent mechanisms. The specific mechanisms are unknown. We will determine how modulation of TLR2 results in activation of PI3K/Akt, leading to cardioprotection. The results of this research will increase our basic science knowledge of the innate immune response to myocardial I/R and how modulation of innate immunity induces cardioprotection. In addition, our data may also have practical significance in that it may lead to the development of new and novel treatment strategies for ischemic heart injury. PUBLIC HEALTH RELEVANCE It is estimated that 500,000 Americans die of heart attacks each year and ischemic heart disease (reduced blood flow in the heart) is responsible for 90% of cardiac mortalities. It is well established that immune responses are involved in the pathogenesis of myocardial ischemia/reperfusion (blood flow again, I/R) injury and heart failure. However, the cellular and molecular mechanisms associated with deleterious immune responses in I/R have not been elucidated. Toll-like receptors (TLRs) are signaling molecules which play a critical role in mediating immune and inflammatory responses. We discovered that TLR4-mediated signaling mediates myocardial I/R injury. Of greater significance, activation of the other molecules (called PI3K/Akt) induces the protection of heart from I/R injury. Thus, our findings indicate that excessive activation of the TLR4-mediated signaling pathway will result in significant heart damage following I/R, while activation of the PI3K/Akt signaling pathway will protect the heart from I/R injury. In this continuation application, we propose to define the cellular and molecular mechanisms by which TLR4 mediates cardiac injury and, more importantly, how modulation of the PI3K/Akt pathway mediates cardioprotection in I/R. We will also define the interplay between TLRs and PI3k/Akt in myocardial I/R. The results of this research will increase our basic science knowledge of the immune response to myocardial I/R and how modulation of immune response induces cardioprotection. Therefore, the work proposed is not only significant but also innovative because our data may also have practical significance in that it may lead to the development of new and novel treatment strategies for heart attack.